RNA interference is a powerful tool to target and silence specific gene expression. The term “RNA interference” (RNAi) was coined after the discovery that injection of double stranded RNA (dsRNA) into C. elegans leads to specific silencing of genes that are highly homologous in sequence to the delivered dsRNA (Fire et al., 1998). RNAi is closely linked to the post-transcriptional gene-silencing (PTGS) mechanism of co-suppression in plants and quelling in fungi (Catalanotto et al., 2000; Cogoni and Macino, 1999; Dalmay et al., 2000, Ketting and Plasterk, 2000; Mourrain et al., 2000; Smardon et al., 2000).
RNAi was discovered when researchers were attempting to use antisense RNA to inactivate a C. elegans gene. The researchers found that injected sense-strand RNA was equally as effective as antisense RNA at inhibiting gene function (Guo et al. (1995) Cell 81: 611-620). Further investigation revealed that the active agent was modest amounts of double-stranded RNA (dsRNA) that contaminated in vitro RNA preparations. Researchers further determined that exon sequences are required and that introns and promoter sequences, while ineffective, did not appear to compromise RNAi.
RNAi can act systemically. This systemic potency was demonstrated by Timmons and Fire (1998 Nature 395: 854). Timmons and Fire performed a simple experiment that produced an astonishing result. They fed to nematodes bacteria that had been engineered to express double-stranded RNA corresponding to the C. elegans unc-22 gene. The transgenic nematodes developed a phenotype similar to that of unc-22 mutants. The results of this and variety of other experiments, in C. elegans and other organisms, indicate that RNAi acts to destabilize cellular RNA after RNA processing.
Double-stranded RNAs (dsRNAs) can provoke gene silencing in numerous in vivo contexts including Drosophila, C. elegans, planaria, hydra, trypanosomes, fungi and plants. Furthermore, short interfering RNA (siRNA), possessing the unique capability to specifically knock down an undesired expression of gene, holds great promises for therapeutics of diversified human diseases. In fact, in it was reported in 2009 that there were 12 ongoing clinical trials using siRNA to treat diseases. Cheng et al., siRNA Delivery and Targeting, Molecular Pharmaceutics, 2009, 6(3):649-650. Of the 12 ongoing trials, 8 trials used naked siRNA for local treatment of ocular and respiratory diseases. Castanotto, et al., The promises and pitfalls of RNA-interference-based therapeutics, Nature, 2009, 457(7228):426-433. In February 2013, there were 28 siRNA clinical trials reported by the National Institute of Health. See National Institute of Health Clinical Trials website. Many of the 28 trials appear to use naked siRNA.
The clinical application of siRNA is constrained by inefficient delivery systems. Specifically, there is a lack of delivery vehicles that are safe, stable, and efficient. To date, various delivery systems have been proposed. Such systems include cationic liposomes, cell-penetrating peptides (CPPs) and cationic polymers. Tseng et al., Lipid-based systemic delivery of siRNA, Advanced Drug Delivery Reviews, 2009, 61(9):721-731 and Lewis et al., Systemic siRNA delivery via hydrodynamic intravascular injection, Advanced Drug Delivery Reviews, 2007, 59(2-3):115-123.
Cationic liposomes and cationic lipids, such as Lipofectamine® and lipid-like materials, are used widely for in vitro studies with high effectiveness; however, the toxicity and low efficiency still restrain their in vivo applications.
For the CPPs-based approaches, RNAi molecules are assembled with CPPs or CPP bioconjugates into complexed particles with significantly improved delivery efficiency. Crombez et al., A New Potent Secondary Amphipathic Cell-penetrating Peptide for siRNA Delivery Into Mammalian Cells, Molecular Therapy, 2009, 17(1):95-103 and Davis et al., Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles, Nature, 2010, 464(7291):1067-U140. Nevertheless, the formation of such assembled structure was driven by weak noncovalent interactions and these particles were generally unstable, particularly against serum nucleases which leads to degradation and poor targeting of the RNAi.
For the cationic-polymer-based approaches, siRNA are assembled with cationic polymers is mainly through the electrostatic interactions. The unique proton sponge effect of the cationic polymers provides the complexes with improved intracellular delivery efficiency. However, similar to the CPPs-based approach, such assembled systems are unstable and readily dissociate and release their siRNA payload before they reach the cytoplasm of the target cells.
Accordingly, in spite of such intensive efforts, the design and synthesis of an effective delivery vehicle for siRNA remains challenging. Thus, there is an ongoing need to develop novel siRNA-delivery methods that are highly robust and effective. Success of this work will provide a general delivery platform with low toxicity and long intracellular half-life for practical therapeutic applications.